CN107533218B

CN107533218B - Method and apparatus for auto-focusing

Info

Publication number: CN107533218B
Application number: CN201680006723.XA
Authority: CN
Inventors: 什洛莫·图尔格曼; 雅艾尔·巴兰; 亚历克斯·埃弗罗斯
Original assignee: IDEA BIO-MEDICAL Ltd
Current assignee: IDEA BIO-MEDICAL Ltd
Priority date: 2015-01-22
Filing date: 2016-01-22
Publication date: 2020-06-12
Anticipated expiration: 2036-01-22
Also published as: EP3248039B1; ES2854700T3; US11092791B2; US20200088980A1; EP3248039A1; US10345566B2; CN107533218A; GB201501093D0; CA3012072C; IL253543A0; GB2534402A; JP2018508837A; IL253543B; CA3012072A1; US20170329115A1; WO2016116897A1; EP3248039A4

Abstract

An autofocus method for determining a focus position of a plurality of wells in at least a portion of a multi-well plate, the method comprising: identifying, using a first objective lens having a first magnification, a focal position of each of the wells relative to the first objective lens in each of at least three wells of the selected subset of the plurality of wells; calculating a plane based on at least three of the focal positions along which the at least three apertures are in focus relative to at least one objective lens having a second magnification that is no greater than the first magnification; and scanning at least some of the plurality of wells in the portion of the plate along the plane using the at least one objective lens.

Description

Method and apparatus for auto-focusing

Technical field and background

The present invention relates generally to the field of optical measurement and/or detection techniques, and more particularly to a method and apparatus for auto-focusing that is particularly useful when viewing non-planar surfaces.

Autofocus is an important feature in many fields of automated detection, such as the computer chip industry, biomedical research, data reading/recording in optical information carriers, etc. In particular, when analyzing samples in multi-well plates comprising a plurality of wells on a single plate, the autofocus of the microscope observing the contents of the wells can make the workflow more efficient, as the operator does not need to focus an objective lens on each well in the plate individually.

In the past, various autofocus methods for inspecting multi-well plates have been disclosed, for example, in U.S. patent No. 7,109,459. However, existing auto-focusing methods may require time-consuming image analysis when using, for example, wells with non-planar bottoms (e.g., multi-well plates with U-shaped bottoms) for growing living cells into spheroids.

Therefore, there is a need for a method for autofocusing a microscope on a multiwell plate that is suitable for multiwell plates having wells with non-planar bottom surfaces.

Disclosure of Invention

There is provided, in accordance with an embodiment of the present invention, an auto-focusing method for determining a focus position of a plurality of wells in at least a portion of a multi-well plate, the method including:

identifying, using a first objective lens having a first magnification, a focal position of each of at least three wells of a selected subset of the plurality of wells relative to the first objective lens in each of the wells;

calculating a plane along which at least three apertures are in focus relative to at least one objective lens having a second magnification that is no greater than the first magnification based on at least three of the focus positions; and

scanning at least some of a plurality of wells in a portion of the plate along the plane using the at least one objective lens.

In some embodiments, the at least one objective lens is a first objective lens, and the first magnification is equal to the second magnification. In some embodiments, calculating the plane comprises calculating a plane along which the at least three apertures are in focus relative to the first objective lens.

In some embodiments, the at least one objective lens is a second objective lens different from the first objective lens, wherein the second magnification is less than the first magnification. In some embodiments, calculating the plane includes translating the at least three focus positions identified using the first objective lens to respective second focus positions relative to the second objective lens based on optical characteristics of the second objective lens; and calculating a plane based on the at least three second focus positions. In some embodiments, computing the plane comprises: calculating a first plane along which at least three apertures are to be focused with respect to the first objective lens, based on at least three of said focus positions; and converting the first plane to a corresponding plane along which the at least three apertures will be focused relative to the second objective based on optical characteristics of the second objective, thereby calculating a plane.

In some embodiments, the at least one objective lens is used for scanning without additional focusing operations.

In some embodiments, the subset of the plurality of wells comprises more than three wells of the plurality of wells.

In some embodiments, identifying the focal position includes identifying the focal position of each aperture in the subset.

In some embodiments, each aperture comprises a substantially cylindrical sidewall and a bottom surface comprising a portion of at least one of a sphere, a paraboloid, and an ellipse. In some embodiments, each aperture has a U-shaped cross-section.

In some embodiments, each aperture includes a generally cylindrical sidewall and a planar bottom surface. In some embodiments, the planar bottom surface is substantially parallel to the top surface of the perforated plate such that the wells have a rectangular cross-section.

In some embodiments, each aperture is frustoconical.

In some embodiments, each aperture has sloped sidewalls, a planar bottom, and a trapezoidal cross-section.

In some embodiments, the method further comprises aligning the first objective lens to be axially over the center of one of the apertures prior to using the first objective lens.

In some embodiments, a portion of the plate comprises one quarter of the plate. In some embodiments, a portion of the plate comprises the entirety of the plate.

There is also provided, in accordance with an embodiment of the present invention, an auto-focusing method for determining a focus position of at least a portion of a hole in a plate, the method including: using a first objective lens having a first magnification to identify a first focal position of at least a portion of the aperture relative to the first objective lens at least one location of the aperture; identifying, for the first focus position, a respective focus position relative to at least one objective lens having a second magnification based on optical characteristics of the at least one objective lens; and scanning at least a portion of the aperture using the at least one objective lens at a height corresponding to the respective focal position, wherein the second magnification is not greater than the first magnification.

In some embodiments, the at least one objective lens is a first objective lens, the first magnification is equal to the second magnification, and the respective focus position is a first focus position.

In some embodiments, the at least one objective lens comprises a second objective lens different from the first objective lens, wherein the second magnification is less than the first magnification. In some such embodiments, the identifying includes translating the first focus position to a corresponding focus position relative to the second objective lens based on an optical characteristic of the second objective lens.

In some embodiments, at least one objective lens is used for scanning without additional focusing operations.

In some embodiments, the aperture comprises a substantially cylindrical sidewall and a bottom surface comprising a portion of at least one of a sphere, a paraboloid, and an ellipse. In some embodiments, the aperture has a U-shaped cross-section.

In some embodiments, the aperture includes a generally cylindrical sidewall and a planar bottom surface. In some embodiments, the planar bottom surface is substantially parallel to the top surface of the plate such that the aperture has a rectangular cross-section.

In some embodiments, the bore is frustoconical. In some embodiments, the holes have sloped sidewalls, a planar bottom, and a trapezoidal cross-section.

There is also provided, in accordance with an embodiment of the present invention, apparatus for automatically determining a focus position of a plurality of wells located in at least a portion of a plate containing wells, the apparatus including: a calculation component programmed to calculate a plane along which at least three apertures in a portion of the plate will be in focus relative to the objective lens; a first objective lens functionally associated with the computing component, the first objective lens having a first magnification, an image from the first objective lens being used by the computing component to identify a focal position for each aperture of at least three apertures of a selected subset of a plurality of apertures; and at least one objective lens having a second magnification that is not greater than the first magnification, the at least one objective lens to scan at least some of a plurality of apertures in a portion of a plate along the plane, wherein the calculation component is configured to calculate a plane based on at least three focus positions, wherein at least three apertures are focused along the calculation plane relative to the at least one objective lens.

In some embodiments, the at least one objective lens is configured to scan a plurality of apertures along a plane without performing additional focusing operations.

In some embodiments, the at least one objective lens is a first objective lens, and the second magnification is equal to the first magnification.

In some embodiments, the at least one objective lens is a second objective lens different from the first objective lens, and the second magnification is less than the first magnification.

In some embodiments, the calculation component is programmed to calculate the focal plane by: translating the at least three focus positions determined using the first objective lens into respective second focus positions relative to the second objective lens based on optical characteristics of the second objective lens; and calculating a focal plane based on the at least three second focal positions.

In some embodiments, the computing component is programmed to compute the focal plane by: calculating a first plane along which at least three apertures are to be focused with respect to the first objective lens based on the at least three focus positions; and converting the first plane to a corresponding plane based on optical characteristics of a second objective lens, the at least three apertures being focused along the corresponding plane relative to the second objective lens, thereby calculating a plane.

In some embodiments, the computing component is programmed to identify a focus position for each aperture in the subset.

In some embodiments, the device is adapted for use with a plate, wherein each aperture comprises a substantially cylindrical sidewall and a bottom surface comprising at least one of a portion of a sphere, a paraboloid, and a portion of an ellipse. In some embodiments, the device is adapted for use with a plate in which each aperture has a U-shaped cross-section.

In some embodiments, the device is adapted for use with a plate in which each well comprises a generally cylindrical side wall and a planar bottom surface. In some embodiments, the planar bottom surface is substantially parallel to the top surface of the plate such that each aperture has a substantially rectangular cross-section.

In some embodiments, the device is adapted for use with plates in which each aperture is frustoconical. In some embodiments, the device is suitable for plates in which each well has an inclined side wall, a planar bottom and a trapezoidal cross-section.

There is also provided, in accordance with an embodiment of the present invention, an autofocus apparatus for automatically determining a focus position of at least a portion of an aperture, the apparatus including: a computing component programmed to compute a focus position of a portion of the aperture; a first objective lens functionally associated with the computing means, the first objective lens having a first magnification, the image from the first objective lens being used by the computing means to identify a focus position of the aperture relative to the first objective lens in at least one position of the aperture; and at least one objective lens having a second magnification that is not greater than the first magnification, the at least one objective lens for scanning at least a portion of the aperture at a height relative to a respective focal position of the aperture of the at least one objective lens, wherein the computing component is programmed to identify the respective focal position based on an optical characteristic of the at least one objective lens.

In some embodiments, the at least one objective lens is configured to scan a portion of the aperture without performing an additional focusing operation.

In some embodiments, the at least one objective lens is a first objective lens, the second magnification is equal to the first magnification, and the respective focus position is the same as the first focus position.

In some embodiments, the at least one objective lens is a second objective lens different from the first objective lens, and wherein the second magnification is less than the first magnification. In some embodiments, the computing component is programmed to identify the respective focus position by translating the first focus position to the respective focus position relative to the second objective lens based on an optical characteristic of the second objective lens.

In some embodiments, the device is adapted for use with an aperture comprising a substantially cylindrical sidewall and a bottom surface comprising at least one of a portion of a sphere, a paraboloid, and a portion of an ellipse. In some embodiments, the device is adapted for use with a bore having a U-shaped cross-section.

In some embodiments, the device is adapted for use with an aperture comprising a substantially cylindrical sidewall and a planar bottom surface. In some embodiments, the planar bottom surface is substantially parallel to the top surface of the plate such that the aperture has a substantially rectangular cross-section.

In some embodiments, the device is adapted for use with a frustoconical bore. In some embodiments, the device is adapted for use with apertures having sloped sidewalls, planar bottoms, and trapezoidal cross-sections.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the terminology (including definitions) of the specification will be prioritized.

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" and grammatical variations thereof are to be taken as specifying the stated features, integers, steps or components, but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms include the terms "consisting of … …" and "consisting essentially of … …".

The indefinite articles "a" and "an" as used herein mean "at least one" or "one or more" unless the context clearly dictates otherwise.

Embodiments of the methods and/or apparatus of the present invention may involve performing or completing selected tasks manually, automatically, or a combination thereof. Some embodiments of the invention are implemented using components comprising hardware, software, firmware or a combination thereof. In some embodiments, some components are general purpose components, such as a general purpose computer or monitor. In some embodiments, some components are application-specific or custom components, such as circuits, integrated circuits, or software.

For example, in some embodiments, some embodiments are implemented as a plurality of software instructions executed by a data processor, e.g., that is part of a general purpose or custom computer. In some embodiments, the data processor or computer includes volatile memory for storing instructions and/or data and/or non-volatile memory for storing instructions and/or data, such as a magnetic hard disk and/or removable media. In some embodiments, the implementation includes a network connection. In some embodiments, implementations include a user interface that generally includes one or more input devices (e.g., to allow commands and/or parameters to be entered) and an output device (e.g., to allow reporting of operating parameters and results).

Drawings

Some embodiments of the invention are described herein with reference to the accompanying drawings. This description together with the drawings make apparent to those skilled in the art how to implement some embodiments of the invention. The drawings are for purposes of illustration and discussion and are not intended to show structural details of the embodiments in more detail than is necessary for a fundamental understanding of the invention. For purposes of clarity, some of the objects depicted in the drawings are not to scale.

In the drawings:

FIGS. 1A and 1B are a top view of a multi-well plate and a cross-sectional view of an individual well in a multi-well plate, the well having a non-planar bottom surface for which embodiments of the present teachings may be useful, respectively;

FIG. 2 is a block diagram of an embodiment of an imaging device for autofocusing samples in a multi-well plate according to an embodiment of the present teachings; and

fig. 3 is a flow chart of an embodiment of a method for automatically focusing an imaging device on a sample in a multi-well plate according to an embodiment of the present teachings.

Detailed Description

The principles, uses and embodiments of the present teachings can be better understood with reference to the accompanying description and drawings. One skilled in the art, upon perusing the description and figures presented herein, will be able to implement the invention without undue effort or experimentation.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the drawings and/or examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. It is also to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Reference is now made to fig. 1A and 1B, which are, respectively, a top view of a multi-well plate and a cross-sectional view of an individual well in a multi-well plate, the well having a non-planar bottom surface for which embodiments of the present teachings may be useful.

As shown in fig. 1A, perforated plate 10 has a top surface 11, side surfaces (not shown), and in some embodiments a bottom surface (not shown). The plate 10 comprises a plurality of holes 12, said holes 12 being arranged in a grid formed by columns 14 and rows 16 and being accessible through holes 17 in the top surface 11. Typically, rows and columns are enumerated or otherwise labeled to enable a user to easily reference a particular well 12. The multi-well plate 10 in the illustrated embodiment contains 96 wells, although other types of plates including, for example, a different number of wells (such as 12, 24, or 384 wells) can also be used with the teachings of the present invention, as described in further detail below. Typically, the holes 12 have a fixed distance between each other and are thus distributed at regular intervals on the plate 10. Specifications regarding the distance between the holes are standard in the art and are also typically provided by the manufacturer of the plate. Typically, the number of holes in the plate has 3: 2, in the longitudinal direction. Thus, the holes may be arranged, for example, in 3 × 2, 6 × 4, 12 × 8, or 24 × 16 grids.

Referring again to FIG. 1B, it can be seen that each hole 12 in the plate 10 can be non-rectangular in cross-section such that the hole has a non-linear bottom surface. In the illustrated embodiment, the well 12 contains a cavity 18 and has a U-shaped cross-section such that the sidewall 20 of the well generally forms a cylinder and the bottom 22 of the well forms a portion of a sphere, a portion of a paraboloid, or a portion of an ellipse, thereby defining a curved floor of the well. Thus, the holes typically have a U-shaped cross-section or a cross-section resembling a paraboloid. Typically, the thickness of the sidewalls 20 and the bottom 22 are the same. An edge 26, which typically forms a portion of and is flush with the top surface 11 of the plate 10 or is raised relative to the top surface 11 of the plate 10, typically surrounds the aperture 12.

Multi-well plates containing wells with non-planar floors are well known in the art and are commercially available from many manufacturers, such as Corning Incorporated Life Sciences of Tewksbury, Mass. Such multiwell plates are used for many types of samples, including for culturing spheroids, culturing non-adherent cells such as lymphocytes and other blood cells, for analysis of 3-dimensional samples, and for processing of compounds. Typically, analysis of these samples requires imaging the sample within the well.

It will be appreciated that since the bottom surface of the well 12 is curved, the area of the observation well which will be focused by the microscope is typically very small and in some cases comprises a single point. Thus, existing autofocus mechanisms, such as those disclosed in U.S. patent No. 7,109,459, are generally not successful in focusing a sample disposed within a well. As described below, the method taught by the present invention enables an operator to automatically focus an imaging device on an aperture having a non-planar bottom, such as the U-shaped aperture 12 in FIG. 1B, without having to manually focus the imaging device on each individual aperture.

It should be understood that although the exemplary schematic shows a hole having a U-shaped cross-section, the methods of the present teachings as described below may be used with other types of holes, such as holes having a planar bottom surface and a rectangular cross-section, or frustoconical holes, i.e., holes that include truncated cones having sloped sidewalls and a planar bottom, and that have a generally trapezoidal cross-section.

Referring now to fig. 2, fig. 2 is a block diagram of an embodiment of an imaging device 200 for autofocusing on wells in a multi-well plate, according to an embodiment of the present teachings.

It should be understood that the present disclosure discusses auto-focusing of wells that include samples by way of example only, and that the same methods and apparatus may also be used for auto-focusing on wells that do not contain samples, or for focusing on multi-well plates in which some wells include samples and others do not.

As shown in fig. 2, imaging device 100 includes a scanning microscope 202 functionally associated with a sample platform movable along X, Y and the Z-axis. The sample platform is configured to have disposed thereon a sample plate 205, which sample plate 205 may be, for example, a plate similar to plate 10 of fig. 1A and 1B.

The microscope 202 also includes a plurality of objective lenses 206 functionally associated with an objective lens changer 208. At any given time, a single objective lens 206 is aligned with a sample platform (not shown) and is operable such that a sample plate disposed on the sample platform may be viewed through the objective lens. The objective lens changer 208 is configured to change the operable lens for observing the specimen when the objective lens needs to be changed. An example of such a converter is described, for example, in WO2012/097191, the contents of which are incorporated herein by reference.

The microscope 202 is functionally associated with at least one illumination source controlled by a control unit (not shown). In some embodiments, the microscope includes a first illumination source comprising a transmissive light source 210a, such as an LED lamp, the transmissive light source 210a configured to illuminate a sample platform during imaging of a sample plate 205 disposed on the sample platform. In some embodiments, the microscope further comprises a second illumination source comprising an excitation light source 210b, the excitation light source 210b being configured to provide illumination to generate a response in a sample loaded on or in the sample plate 205, such as to provide illumination to excite a fluorescent or luminescent component of the sample. In some embodiments, illumination from light sources 210a and/or 210b impinges on one or more optical elements 212, such as mirrors, dichroic cubes, beam splitters, filters, and the like, prior to impinging on a sample disposed on sample plate 205. In some embodiments, illumination from illumination source 210 passes through optical fiber 213 before striking the sample.

In some embodiments, the image viewable through the microscope 202 is acquired by an image acquisition unit (not shown) and transferred to the processing unit 214 for further processing and analysis.

Referring now to fig. 3, fig. 3 is a flow chart of an embodiment of a method for automatically focusing an imaging device on a sample in a multi-well plate according to an embodiment of the present teachings.

The methods described below may be used in an imaging device, such as imaging device 200 of fig. 2, to automatically determine the focus position of a plurality of samples disposed in a sample plate, such as plate 10 of fig. 1A, that contains a plurality of wells. The method may be performed on plates containing wells having non-planar bottom surfaces, such as well 12 of fig. 1B, or on other types of wells, such as wells having planar bottom surfaces or frustoconical wells having sloped sidewalls and planar bottoms, etc.

A subset of the holes in the plate is selected, as shown in step 300. In some embodiments, the subset includes at least three wells each containing a liquid or sample, although this is not necessary for the methods disclosed herein. At step 302, for at least three apertures in the subset, and in some embodiments for all apertures in the subset, for a first objective lens having a first magnification (e.g., objective lens 206 of FIG. 2), a focus position of a sample contained in the aperture is identified.

Typically, the first objective lens has a considerable magnification, such as 20x, 10x, etc.

In some embodiments, the subset contains more than three apertures, but the focus positions are identified only for three apertures in the subset. In some embodiments, the subset contains more than three apertures, and the focal positions are identified for more than three apertures in the subset, but not for all apertures in the subset. For example, the subset may contain at least five apertures, and the focal positions are identified for at least four apertures in the subset, rather than for all apertures. In some embodiments, the focal position is identified for all of the apertures in the subset.

The focal position of the sample in the wells of the subset may be identified using any suitable method known in the art, including manual and automated methods. In some embodiments, the focus position is identified substantially as described in U.S. patent No. 7,109,459, the contents of which are incorporated herein by reference as if fully set forth herein.

According to the teaching of us patent No. 7,109,459, to determine the focus position, the focal plane of the first objective lens is spaced from a surface of the plate, such as the bottom surface of the plate, by a distance, for example, about one millimeter. The focal plane of the objective lens is then shifted towards the plate, for example by shifting the objective lens or the plate relative to each other. For example, the objective lens may be arranged below the plate such that the focal plane of the objective lens is below the surface of the plate and the focal plane is displaced vertically upwards towards the surface of the plate.

During the shift of the focal plane of the objective lens, the control hardware of the microscope records the intensity of the light reflected off the plate until the intensity of the detected light reaches a maximum value, which in some embodiments is above a preset threshold. This maximum value of the detected light intensity corresponds to the focal position of the surface of the plate.

Without wishing to be bound by theory, in the above example, in which the objective lens is arranged below the plate and the focal plane is initially set below the plate and displaced towards the plate, the location at which the maximum light intensity is observed is considered to correspond to the point at which the focal plane of the objective lens is tangent to the curved surface of the aperture bottom.

Subsequently, in some embodiments, the focal plane of the objective lens continues to be displaced towards the plate until another peak in the reflected light intensity is detected, which peak is defined by a respective threshold value depending on the environment and the sample being detected. Without wishing to be bound by theory, in the above example, where the objective lens is arranged below the plate and the focal plane is initially disposed below the plate and displaced towards the plate, it is believed that a second peak in reflected light intensity occurs when the focal plane of the objective lens is tangent to the bottom of the well plate, and that this second peak generally represents a shift from the focus position of the sample. The size of the offset may be determined manually by a user or may be determined automatically using methods known in the art.

In some embodiments, the offset is calculated from a first peak in the intensity of the detected light without continuing to search for a second peak in the intensity of the detected light. In such embodiments, the size of the offset may be determined manually by a user, or may be determined automatically using methods known in the art.

It will be appreciated that the direction in which the focal plane is displaced towards the plate and the order in which the peaks in the intensity of the detected light are identified depends on the settings of the imaging device. For example, in some embodiments, the objective lens is arranged below the sample plate, but the focal plane of the objective lens is disposed above the bottom of the well such that the focal plane is displaced downward towards the bottom of the well. Without wishing to be bound by theory, in these embodiments it is believed that a first peak in reflected light intensity occurs when the focal plane of the objective lens is tangent to the bottom of the well plate, and that this first peak generally represents a shift from the focused position of the sample, while the position of a second peak in detected reflected light intensity corresponds to a point where the focal plane of the objective lens is tangent to the curved surface of the well bottom. A corresponding situation may also arise in other embodiments, in which the objective lens is arranged above the sample plate and the focal plane of the objective lens is arranged above the bottom of the well, such that the focal plane is displaced downwards towards the bottom of the well.

As another example, in some embodiments the objective lens is arranged above the sample plate, but the focal plane of the objective lens is arranged below the bottom of the well, such that the focal plane is displaced upwards towards the bottom of the well. Without wishing to be bound by theory, in these embodiments it is believed that the position of the first peak in the detected reflected light intensity corresponds to the point at which the focal plane of the objective lens is tangent to the curved surface of the well bottom, whereas a second peak in the reflected light intensity occurs when the focal plane of the objective lens is tangent to the well plate bottom, and this second peak generally represents a shift from the focused position of the sample.

In some embodiments, the center of the hole where the focal position is located is determined based on the specifications of the plate provided by the manufacturer. In some embodiments, the center of the aperture is determined using an X-Y displacement of the plate or an X-Y displacement of the objective lens until the center of the aperture or the edge of the aperture is determined using suitable light detection parameters and features, as is known in the art.

At step 304, at least three focal positions determined in step 302 are used to calculate a plane along which at least some, and typically all, of the plurality of apertures in the plate are focused or nearly focused with respect to the second objective lens (such as objective lens 206 of FIG. 2). The second objective lens has a second magnification that is not greater than the first magnification of the first objective lens. As described below, the second objective lens is used to scan the aperture based on the location calculation plane, by maintaining the position of the second objective lens during scanning such that the calculated plane and the focal plane of the second objective lens are coincident or nearly coincident for any given scanned aperture.

In some embodiments, the plane is calculated by: at least three (typically each) of the focal positions determined in step 302 using the first objective lens are translated into respective second focal positions relative to the second objective lens based on the optical characteristics of the second objective lens, and a plane including the at least three second focal positions is calculated.

In some embodiments, the plane is calculated by: calculating a first plane based on at least three focal positions, wherein along the first plane at least some of the plurality of wells in the plate, and typically all of the wells in the plate, are in focus or near focus with respect to the first objective lens; the first plane is then converted into a corresponding plane based on the optical characteristics of the second objective lens, along which at least some of the apertures, and typically all of the apertures, are focused or nearly focused with respect to the second objective lens.

As described above, the second objective lens has a magnification not larger than the first magnification of the first objective lens. Thus, in some embodiments, the second magnification is less than the first magnification, and may be, for example, 4x or 2 x. In some embodiments, the second magnification is equal to the first magnification, but the second objective lens has a numerical aperture value that is higher than the numerical aperture value of the first objective lens.

In some embodiments, the plane is calculated using all of the focus positions identified in step 302. In other embodiments, the plane is calculated using less than all of the focus positions identified in step 302.

In some embodiments, for a portion of the plate (e.g., a quarter of the plate), the plane is calculated using at least three focal positions identified with the first objective lens within that portion of the plate. In such embodiments, for each portion or each quarter of the plate, the method described herein is repeated using a different set of focus positions for each such portion.

At step 306, which may occur before or after step 304 described above, the first objective lens is replaced with the second objective lens, for example, by a suitable hardware mechanism (such as objective lens changer 208 of fig. 2). In some embodiments, the first and second objective lenses are identical, thereby eliminating step 306 of FIG. 3.

Finally, at step 308, the second objective lens is used to scan or image the wells of the multi-well plate along the plane calculated in step 304 without any additional focusing operations.

The scanning of step 308 may be performed using any suitable method known in the art, including capturing a stack of images, which is particularly useful when imaging three-dimensional structures such as spheroids. In some embodiments, the teachings herein can be implemented on a plate having a single well or on a single well within a multi-well plate. In these embodiments, a first objective lens is used to find the focal point of the sample in the plate. Based on the optical characteristics of the second objective lens, the focal point found using the first objective lens is converted into the focal point of the second objective lens. Then, when the second objective lens is placed at the translated focus point or at its height, the second objective lens is used to scan the plate.

It should be appreciated that the teachings herein allow for focusing of the imaging device relative to the plate, regardless of "expected height differences" and "unexpected height differences" within the tube plate. The "expected height difference" is defined as the curvature of the plate listed in the specification of the plate and is the curvature that the manufacturer wishes to have in the plate, for example having a curved bottom due to the structure. An "unexpected height difference" is defined as a lack of flatness, which is undesirable in the specifications of the panel. The "unexpected height difference" may be, for example, due to a difference in the relative heights of the bottoms of the wells; or may be a deviation of flatness in a virtual surface tracked by the scanning component, e.g. as the objective lens is moved; or the surface on which the plate rests is not parallel to the virtual surface tracked by the scanning component as the objective lens moves.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the included claims.

Any reference cited or identified in this application is not to be construed as an admission that such reference is available as prior art to the present invention.

Section headings are used herein for simplicity of understanding the specification and should not be construed as necessarily limiting.

Claims

1. An auto-focusing method for determining a focus position of a plurality of wells in at least a portion of a multi-well plate, the method comprising:

identifying, using a first objective lens having a first magnification, a focal position of each of the wells relative to the first objective lens in each of at least three wells of the selected subset of the plurality of wells;

calculating a plane based on at least three of the focal positions along which the at least three apertures are focused relative to a second objective lens having a second magnification that is less than the first magnification; and

scanning at least some of the plurality of holes in the portion of the plate along the plane using the second objective lens.

2. The auto-focusing method of claim 1, wherein the calculating a plane comprises:

translating at least three of the focus positions identified using the first objective lens into respective second focus positions relative to the second objective lens based on optical characteristics of the second objective lens; and

calculating the plane based on at least three of the second focus positions.

3. The auto-focusing method of claim 2, wherein the calculating a plane comprises:

calculating a first plane based on at least three of the focal positions, wherein the at least three apertures are focused along the first plane relative to the first objective lens; and

calculating the plane by converting the first plane to a corresponding plane along which the at least three apertures are focused relative to the second objective lens based on optical characteristics of the second objective lens.

4. The auto-focusing method according to any one of claims 1 to 3, wherein the scanning is performed using the second objective lens without performing an additional focusing operation.

5. The autofocus method of any of claims 1 to 3, wherein the subset of the plurality of wells comprises more than three wells of the plurality of wells.

6. The auto-focusing method of any one of claims 1 to 3, wherein said identifying a focus position comprises identifying the focus position for each aperture in the subset.

7. The auto-focusing method of any one of claims 1 to 3, wherein each of the holes comprises a substantially cylindrical sidewall and a bottom surface containing a portion of at least one of a sphere, a paraboloid, and an ellipse.

8. The auto-focusing method of claim 7, wherein each of the holes has a U-shaped cross-section.

9. The autofocus method of any of claims 1 to 3, wherein each of the wells comprises a substantially cylindrical sidewall and a planar bottom surface.

10. The autofocus method of claim 9, wherein the bottom surface of the plane is substantially parallel to the top surface of the multiwell plate, such that the well has a rectangular cross-section.

11. The autofocus method of any of claims 1 to 3, wherein each of the holes is frustoconical.

12. The auto-focusing method according to any one of claims 1 to 3, wherein each of the holes has an inclined sidewall, a planar bottom, and a trapezoidal cross section.

13. The autofocus method of any of claims 1 to 3, further comprising aligning the first objective lens to be axially over a center of one of the apertures prior to using the first objective lens.

14. The autofocus method of any of claims 1 to 3, wherein the portion of the plate comprises a quarter of the plate.

15. The auto-focusing method according to any one of claims 1 to 3, wherein the portion of the plate includes all of the plate.

16. An autofocus apparatus for automatically determining the focus position of a plurality of wells located in at least a portion of a plate containing wells, the apparatus comprising:

a computing component programmed to compute a plane along which at least three apertures in the portion of the plate are focused relative to an objective lens;

a first objective lens functionally associated with the computing component, the first objective lens having a first magnification, an image from the first objective lens being used by the computing component to identify a focus position for each aperture of at least three apertures of the selected subset of the plurality of apertures; and

a second objective having a second magnification that is less than the first magnification, the second objective for scanning at least some of the plurality of apertures in the portion of the plate along the plane,

wherein the calculation component is configured to calculate the plane based on at least three of the focus positions along which the at least three apertures are focused relative to the second objective lens.

17. The autofocus device of claim 16, wherein the second objective lens is configured to scan the plurality of apertures along the plane without performing an additional focusing operation.

18. The autofocus device of claim 16, wherein the computing component is programmed to compute the focal plane by:

calculating the focal plane based on at least three of the second focal positions.

19. The autofocus device of claim 16, wherein the computing component is programmed to compute the focal plane by:

20. An autofocus device according to any one of claims 16 to 19, wherein the computing means is programmed to identify the focus position for each aperture in the subset.

21. An autofocus device according to any one of claims 16 to 19, wherein the device is adapted for use with a plate, wherein each aperture in the plate comprises a substantially cylindrical side wall and a base comprising at least one of a portion of a sphere, a paraboloid and a portion of an ellipse.

22. An autofocus device according to any of claims 16 to 19, wherein the device is adapted for use with a plate in which each aperture has a U-shaped cross-section.

23. An autofocus device according to any of claims 16 to 19, wherein the device is adapted for use with a plate in which each aperture comprises a substantially cylindrical side wall and a planar base.

24. The autofocus device of claim 23, wherein the bottom surface of the plane is substantially parallel to the top surface of the plate, such that each of the holes has a substantially rectangular cross-section.

25. An autofocus device according to any one of claims 16 to 19, wherein the device is adapted for use with a plate in which each of the apertures is frusto-conical.

26. An autofocus device according to any one of claims 16 to 19, wherein the device is adapted for use with a plate in which each aperture has inclined side walls, a planar base and a trapezoidal cross-section.

27. The autofocus device of any of claims 16 to 19, wherein the portion of the plate comprises a quarter of the plate.

28. The autofocus device of any of claims 16 to 19, wherein the portion of the plate comprises all of the plate.